The overall goal of this RENEWAL proposal is to analyze the molecular mechanism by which capillary endothelial (CE) cell growth is regulated by local changes in extracellular matrix (ECM) structure. The working hypothesis that has driven this effort for the past 9 years is that ECM molecules control CE cell cycle progression in the presence of soluble angiogenic mitogens by resisting tensional forces generated in the cytoskeleton (CSK) that are exerted on integrin receptors, and thereby promoting changes in cell shape and cytoskeletal structure. In past grant periods, we confirmed that the intact actin CSK of spread CE cells conveys specific signals during a discrete period in mid G1 phase that are critical for upregulation of cyclin D1 and suppression of p27, and hence for passage through the late GIlS check-point. Our recent work has revealed that small Rho family GTPases, notably RhoA, may link cell spreading and associated CSK changes to the cell cycle machinery through activation of its downstream effector mDia and modulation of the F-box protein Skp2 that targets p27 for degradation. This proposal focuses on two fundamental questions: how do structural changes in the CSK associated with cell spreading on ECM activate Rho signaling? and how does activated Rho regulate the cell cycle machinery? Preliminary results show that the level of isometric tension in the CSK feeds back to regulate Rho activity, suggesting that changes in the cytoskeletal force balance that occur during cell spreading may be responsible for control of Rho activity during mid to late G1. The main goal of this 5 year continuation proposal is therefore to answer these fundamental questions by extending our ongoing studies with human CE cells and pursuing the following specific aims: 1) To determine how integrin engagement, stress application to integrins, and cell shape distortion contribute to the activation of RhoA, 2) To analyze how Rho GTPases increase Skp2 and thereby promote cell shape-dependent progression through the GI/S transition, and 3) To explore how mDia contributes to Rho dependent control of cell cycle progression. Cell shape will be controlled using substrates coated with different densities of ECM molecules, microfabricated ECM islands that exhibit defined size and geometry on the micron scale, and flexible ECM substrates that differ in their mechanical compliance. Endogenous changes in cytoskeletal tension will be quantitated using traction force microscopy. Exogenous stresses will be applied to the integrins using RGD-coated magnetic microbeads in conjunction with applied magnetic fields. This experimental approach will enable us to translate the long recognized coupling between cell shape and growth into molecular terms. It also may lead to identification of novel molecular targets for angiogenesis inhibition and hence, new approaches to anti-cancer therapy.